摘要
为检测母猪产前血清和初乳中的PEDV IgA抗体水平,并分析二者的相关性,本研究利用真核表达的PEDV S1蛋白作为包被抗原,HRP标记的羊抗猪IgA作为酶标二抗,建立PEDV S1 IgA抗体间接ELISA(S1-iELISA)方法,并对反应条件进行优化。结果显示,该方法中最佳抗原包被量为50 ng/孔,酶标二抗(IgA-HRP)1:10000稀释,血清或乳汁样品1:50稀释,临界值为0.207,初步建立了PEDV S1-iELISA方法。利用该方法检测PEDV、猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪伪狂犬病毒(PRV)、猪圆环病毒2型(PCV2)的阳性血清,结果显示,除PEDV阳性血清外,其他病原血清均为阴性,该方法特异性强。将猪感染野毒后的血清和乳汁分别稀释至1:12800和1:512000时,以及猪免疫疫苗后的血清和乳汁分别稀释至1:800和1:2000时,检测结果仍为阳性,该方法的敏感性较高。分别采用同一批次抗原包被板和不同批次的抗原包被板分别进行3次重复检测,结果显示批内和批间重复性试验的平均变异系数均小于5%,重复性好。利用IDEXX PEDV IgA检测试剂盒和本实验建立的方法同时检测188份临床血清样品,结果显示,二者阴性符合率为96.7%,阳性符合率为97.9%。利用该方法对不同免疫背景的5个猪场母猪产前多个时间点血清和初乳中的IgA抗体水平检测,并分析二者相关性,结果显示,母猪产前血清和初乳中的IgA抗体水平具有较强的正相关性。本研究建立的PEDV S1-iELISA方法优于商品化检测试剂盒,基于该方法检测母猪产前血清中的IgA抗体水平,可提前预测其初乳中的IgA抗体水平,有利于母猪PEDV疫苗免疫效果的评价及免疫程序的及时调整,为提高初乳中的PEDV抗体水平赢得时间,从而最大可能保证新生仔猪获得高水平的PEDV IgA抗体,降低PEDV感染风险及损失,对PED的防控具有重要指导意义。
In order to detect the level of PEDV IgA antibody in prenatal serum and colostrum of sows and analyze its correlation,this study used eukaryotic expressed PEDV S1 protein as the coating antigen and HRP-labeled goat anti-pig IgA as the enzyme-labeled secondary antibody to optimize the reaction conditions.The results showed that the optimal antigen coating amount in this method was 50ng/well,the enzyme-labeled secondary antibody was diluted 110000,the serum or colostrum samples were diluted 150,and the cutoff value was 0.207.An indirect ELISA method for PEDV S1 IgA antibody(S1-iELISA)detection was preliminarily established.The method was used to detect the positive serum of PEDV,CSFV,PRRSV,PRV and PCV2.The results showed that except for the PEDV positive serum,the serum of other pathogens were negative,indicating that the method was specific.When the serum and colostrum of pigs infected with wild virus were diluted to 112800 and 1512000 respectively,and the serum and colostrum of pigs immunized with vaccine were diluted to 1800 and 12000 respectively,the test results were still positive,suggesting the method had high sensitivity.The results of intra-batch and inter-batch analysis showed that the average coefficient of variation of intra-batch and inter-batch was less than 5%,indicating that the repeatability of this method was good.The IDEXX PEDV IgA detection kit and the method established in this experiment were used to detect clinical samples.The results showed that the negative coincidence rate was 96.7%,and the positive coincidence rate was 97.9%.In this study,this S1-iELISA method was used to detect the IgA antibody levels in the serum and colostrum of sows from five pig farms with different immune backgrounds at multiple time points before delivery,and the correlation between the two was analyzed.The results showed that there was a strong positive correlation between the IgA antibody levels in serum and colostrum of sows.The PEDV IgA iELISA method established in this study is superior to the commercial detection kit.Based on this method,the IgA antibody level in the prenatal serum of sows can be predicted in advance,which is beneficial to the evaluation of the immune effect of PEDV vaccine in sows and adjustment of the immunization procedure in time to increase the PEDV antibody level in the colostrum.This can ensure that the newborn piglets obtain high levels of PEDV IgA antibodies,reduce infection risks and losses,and has important guiding significance for the prevention and control of PED.
作者
邓瑞德
陈志雄
胡泽奇
李润成
王亚
蒋敏
颜运秋
龚自冶
董伟
葛猛
DENG Rui-de;CHEN Zhi-xiong;HU Ze-qi;LI Run-cheng;WANG Ya;JIANG Min;YAN Yun-qiu;GONG Zi-ye;DONG Wei;GE Meng(College of Veterinary Medicine,Hunan Agricultural University,Changsha 410128,China;Hunan Jinghong Agriculture and Animal Husbandry Co.,Ltd.,Chaling 412400,China;Hunan Tianxin Breeding Share Co.,Ltd.,Changsha 410029,China)
出处
《中国预防兽医学报》
CSCD
北大核心
2024年第12期1245-1254,共10页
Chinese Journal of Preventive Veterinary Medicine
基金
湖南省重点研发计划项目(2023NK2017)
国家自然科学基金面上项目(32072871)
云南省重大科技专项计划(202202AE090032)。
